子宫内膜中TLR4的定位及pcDNA3.1-TLR4载体的构建
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摘要
试验旨在研究TLR4(Toll like receptor 4)蛋白在子宫内膜组织中具体的分布与表达,从而构建TLR4真核表达载体,为TLR4在细胞水平上的功能研究提供一种技术手段。选择早期妊娠绵羊(9 d、13 d、17 d、21 d、25 d)子宫组织,采用免疫组织化学和细胞免疫学抗体着色法检测各阶段子宫组织中TLR4蛋白的分布与表达,确定受体蛋白的表达部位。同时将构建的pcDNA3.1-TLR4质粒转染到确定的受体细胞,检测TLR4蛋白的表达水平。结果表明:TLR4蛋白在子宫内膜各部位均有表达,呈一定的动态时空模式,且第17 d胚胎附植后主要表达于内膜基质细胞;重组质粒转染到基质细胞后,TLR4蛋白的表达水平明显增强。此研究成功构建pcDNA3.1-TLR4表达载体,为TLR4在生殖细胞免疫学功能的研究奠定了一定的理论基础。
To study the specific distribution and expression of TLR4 protein in endometrium, and to construct a TLR4 eukaryotic expression vector and provide a technical means for the study of the function of TLR4 at the cell level,the uterine tissues of the early pregnant sheep(9 d, 13 d, 17 d, 21 d, 25 d) were selected. The distribution and expression of TLR4 protein in the uterus tissues of all stages were detected by immunohistochemistry and immunological antibody coloring, and the expression of the receptor protein was determined. The constructed pcDNA3.1(+)-TLR4 plasmid was transfected into the identified receptor cells, and the expression level of TLR4 protein was detected.TLR4 protein was expressed in all parts of endometrium, showing a dynamic spatial and temporal pattern. Moreover, endometrial stromal cells were mainly expressed after implantation of 17 d. After transfection of recombinant plasmid into stromal cells, the expression level of TLR4 protein was significantly enhanced.pcDNA3.1(+)-TLR4 expression vector was successfully constructed, which laid a theoretical foundation for TLR4 in the study of germ cell immunology function.
To study the specific distribution and expression of TLR4 protein in endometrium, and to construct a TLR4 eukaryotic expression vector and provide a technical means for the study of the function of TLR4 at the cell level,the uterine tissues of the early pregnant sheep(9 d, 13 d, 17 d, 21 d, 25 d) were selected. The distribution and expression of TLR4 protein in the uterus tissues of all stages were detected by immunohistochemistry and immunological antibody coloring, and the expression of the receptor protein was determined. The constructed pcDNA3.1(+)-TLR4 plasmid was transfected into the identified receptor cells, and the expression level of TLR4 protein was detected.TLR4 protein was expressed in all parts of endometrium, showing a dynamic spatial and temporal pattern. Moreover, endometrial stromal cells were mainly expressed after implantation of 17 d. After transfection of recombinant plasmid into stromal cells, the expression level of TLR4 protein was significantly enhanced.pcDNA3.1(+)-TLR4 expression vector was successfully constructed, which laid a theoretical foundation for TLR4 in the study of germ cell immunology function.
引文
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[23] 杜玉梅,左正宏.基因功能研究方法的新进展[J].生命科学,2008,20(4):589-592.
[2] ANDERSON K V,BOKLA L,NUSSLEIN V.C,et al.Establishment of dorsal-ventral polarity in the Drosophila embryo:the induction of polarity by the Toll gene product[J].Cell,1985,42(3):779-798.
[3] JANEWAY A.Approaching the asymptote Evolution and revolution in immunology[J].Cold Spring Harb Syrup Quant Biol,1989,54(1):1-13.
[4] MEANS T K,GOLENBOCK D T,FENTON M J,et al.Structure and function of Toll-like receptor proteins[J].Life Sciences,2000,68(3):241-258.
[5] Huang B,Zhao J,UNKELESS J C,et al.TLR signaling by tumor and immune cells:a double-edged sword [J].Oncogene,2008,27(2):218-224.
[6] DOYLE S L,JEFFERIES C A,et al.Bruton′s tyrosine kinase is involved in p65-mediated transactivation and phosphorylation of p65 on serine 536 during NF-kB activation by lipopolysaccharide[J].Journal Biological Chemistry,2005,280(25):23 496-23 501.
[7] FU H Y,LI C,YANG W,et al.FOXP3 and TLR4 protein expression are correlated in non-small cell lung cancer:implications for tumor progression and escape[J].Acta Histochemica,2013,115(2):151-157.
[8] 满晓华,孙运良,龚燕芳,等.TLR4在胰腺癌组织中的表达及其与肿瘤血管生成的关系[J].中华胰腺病杂志,2012,12(3):167-169.
[9] YUAN X,ZHOU Y,WANG W,et al.Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production[J].Cell Death Disease,2013,4(9):1-10.
[10] RAKOFF N S,MEDZHITOV R.Toll-like receptors and cancer[J].Nature Reviews Cancer,2009,9(1):57-63.
[11] 孔维欢.TLR4基因在绵羊胚胎附植期子宫内膜的表达模式研究[D].石河子:石河子大学,2018.
[12] 熊燊源.ATF4在绵羊早期妊娠子宫和体外受精胚胎的表达及功能研究[D].石河子:石河子大学,2017.
[13] 张明.牛IFN-τ基因成熟蛋白CDS区的克隆,原核表达及其在妊娠建立过程中的生物学功能研究[D].雅安:四川农业大学,2007.
[14] GUO J,CHEN L,LUO N,et al.LPS/TLR4-mediated stromal cells acquire an invasive phenotype and are implicated in the pathogenesis of adenomyosis[J].Scientific Reports,2016,6:21 416.
[15] 张金凤.牛/FNT基因的功能研究及TEAD家族和YAP1基因对其表达的影响[D].哈尔滨:东北农业大学,2013.
[16] SPENCER T E,GREG A J,FULLER W,et al.Implantation mechanisms:insights from the sheep[J].Reproduction,2004,128(6):657-668.
[17] ZHANG Y L,DAVIS D L.Morphology of luminal and glandular epithelial cells from pig endometrium grown on plastic or extracellular matrices[J].Journal of Animal Science,2000,78(1):131-138.
[18] BLITEK A,ZIECIK A J.Prostaglandins f and e secretion by porcine epithelial and stromal endometrial cells on different days of the oestrous cycle[J].Reproduction in Domestic Animals,2004,39(5):340.
[19] 字友梅,王少元.新基因功能的研究策略[J].医学综述,2011,17(10):1 478-1 481.
[20] PANTS A G,BARBER M R,WOOD M J,et al.Establishment and characterization of a caprine mammary myoepithelial cell line (CMMyoEC)[J].Vitro Cellular & Developmental Biology Animal,2000,36(1):351-356.
[21] 卜友泉,杨正梅,宋方洲,等.新基因功能研究的策略与方法[J].生命科学研究,2006,(1):96-99.
[22] 孙春晓,于常海.基因功能研究的技术和方法[J].医学分子生物学杂志,2000,(2):112-115.
[23] 杜玉梅,左正宏.基因功能研究方法的新进展[J].生命科学,2008,20(4):589-592.